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878. The T1α Promoter Mediates Nuclear Import of Plasmid DNA into Alveolar Epithelial Type I Cells
LUNG AND RESPIRATORY DISEASE GENE & CELL THERAPY 878. The T1Į Promoter Mediates Nuclear Import of Plasmid DNA into Alveolar Epithelial Type I Cells Lynn F. Gottfried, David A. Dean. Pediatrics, University of Rochester Medical Center, Rochester, NY.
Nonviral gene delivery to the lung is a promising approach for the treatment of a various number of devastating diseases. However, its use has been limited, in part, by the lack of cell specic targeting of DNA. The alveolar epithelial type I (ATI) cell, in particular, is an attractive cell type to target, as it comprises 95% of the internal surface area of the lung. However, ATI-specific gene delivery studies have been limited due to a lack of understanding of the true transcriptional properties of these cells and difculty in isolation of primary cell cultures. To overcome this, we characterized two models of ATI cells, the R3/1 cell line and primary rat alveolar type II cells maintained on plastic for 5 days, to be used for initial gene delivery studies. Immunouorescence analysis has identied these cell types as good models for ATI cells, as evidenced by their strong expression of the ATI markers, T1α and Aquaporin-5, and absent expression of ATII markers, Surfactant Protein B and Lamellar Body Membrane 180. Using these models, we have developed a method for ATI-specic targeting of plasmid DNA through utilization of cellspecic DNA nuclear import sequences. We have previously shown that nuclear import of plasmids in non-dividing cells is mediated by specic sequences of DNA that bind to transcription factors in the cytoplasm. These transcription factors contain nuclear localization signals that can be presented to the importin nuclear import machinery to promote nuclear import. If bound to the transcription factors, the DNA can be carried into the nucleus. To date, we have identied DNA sequences that act in all cell types as well as specic cell types, due to the transcription factors bound. We screened the promoters from several genes previously identied in the literature to be specic for or enriched in ATI cells for the ability to mediate DNA nuclear import in this specic cell type after cytoplasmic microinjection. The promoters of these genes were inserted into a plasmid lacking any other DNA nuclear targeting sequence, and the plasmids were labeled with a triplex-forming Cy3-PNA. Of the sequences examined, we demonstrate that only a DNA sequence within the rat T1α promoter was able to mediate plasmid DNA nuclear import in R3/1 and day 5 primary rat alveolar epithelial cells. When microinjected into other cell types, the T1α promoter displayed no DNA nuclear import activity, suggesting that this sequence acts in a type I cell-specic manner. This highlights a new property of the T1α promoter as well as the ability to directly target the ATI cell over other cell types of the lung.
879. Effects of LPC on Lung Mechanics in NonHuman Primates P. Hiatt, R. McConnell, N. Grove, D. Dang, N. Brunetti-Pierri, D. Palmer, A. Beaudet, P. Ng. Depts. of Pulmonary Pediatrics and Molecular Human Genetics, Baylor College of Medicine, Houston, TX.
Efcient transduction of the airway epithelium and long-term expression of the transgene are essential elements for successful Cystic Fibrosis (CF) gene therapy. Parsons et al (2008) showed that lysophosphatidylcholine (LPC) opens tight junctions between cells allowing improved access to viral receptors on the baso-lateral surface of the airway epithelium in mice. We have previously shown that targeted lobar aerosolization of a mixture of LPC and Helper Dependent Adenovirus (HDAd) into nonhuman primate lungs results in uniform, high level pulmonary transduction. Our goal is to use LPC as a carrier in the delivery of HDAd for gene therapy in CF patients. In this study we investigated the effects of LPC on lung mechanics of non-human primates. Methods: Baseline pulmonary function (PF) in 6 baboons was measured using the Flexivent respiratory mechanics platform (Scireq). Animals were intubated with a cuffed endotracheal Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
tube, placed under general anesthesia, and their lungs were inated to 30cm of H2O prior to each of the PF measurements. Following baseline measurements, LPC was aerosolized in two different concentrations which were previously shown to yield signicant pulmonary transduction by HDAd. Two ml/lobe of 0.1% LPC was delivered into each of the six major lung lobes of baboons using an intracorporeal nebulizing catheter (AeroProbe) inserted into a bronchoscope. PF measurements were taken after LPC administration and every 15 minutes thereafter up to 2.5 hours to determine changes in lung mechanics. A nal PF measurement was taken at 24 hours post LPC administration for those animals with lung functions that did not return to baseline at the end of the 2.5 hour testing time. Three weeks after administration of 0.1% LPC, 0.05% LPC was administered to the same animals and PF measurements were obtained as previously described. Results: Maximal PF changes occurred 15 minutes following LPC administration for both 0.05% and 0.1%LPC. Mean respiratory resistance increased 53%+14%(SE) and 44%+14% above baseline and compliance decreased 25%+3.9% and 27%+7% below baseline after the delivery of 0.05% LPC(n=6) and 0.05%LPC+vector(n=3), respectively. Respiratory resistance in these animals returned to normal in less than 3 hours. When 0.1% LPC(n=5) was administered to the same baboons, their mean respiratory resistance increased 127%+32% over baseline and compliance decreased 35%+4.1% from baseline. When 0.1%LPC+vector was administered to 3 baboons, their mean respiratory resistance increased 338%+108% over baseline and compliance decreased 63%+4.2% from baseline. Pulmonary function changes returned to baseline within 24 hours of LPC+vector administration. No significant changes in FiO2 or SpO2 were observed following administration of either concentrations of LPC. Conclusion: LPC (0.05% and 0.1%) is associated with increased respiratory resistance and decreased compliance measurements when aerosolized to the lung. Maximal changes in PF occur within 15 minutes of delivery. These results indicate that LPC delivered by aerosol to the airways of the lung produce signicant but temporary pulmonary function changes in non-human primates in a dose-dependent manner.
880. Repeat Aerosol Delivery of Concentrated PEI/pDNA to the Sheep Lung
Gerry McLachlan,1 Lee A. Davies,2 Cath M. Gordon,1 Christina Vrettou,1 Eilidh Baker,1 Peter Tennant,1 Alison Baker,1 Rebecca L. Coles,2 Dominique McCormick,2 Stephanie G. Sumner-Jones,2 Stephen C. Hyde,2 Deborah R. Gill,2 D. David S. Collie.2 1 The Roslin Institute & R(D)SVS, University of Edinburgh, Edinburgh, United Kingdom; 2Gene Medicine Group, NDCLS, University of Oxford, Oxford, United Kingdom. Considerable advances have been made in lung-directed gene therapy, however it is unlikely that clinical benet to patients with chronic lung disease can be achieved without repeat administration. For viral vectors, the ability to deliver multiple doses is normally limited by the host immune response, which results in attenuated expression following consecutive exposures. Importantly, aerosol delivery of non-viral gene transfer agents to the lung is not generally associated with the same problem. We have previously demonstrated that aerosol delivery of plasmid DNA (pDNA) complexed with polyethylenimine (PEI) to sheep, either at the standard (low) concentration or with a novel concentrated formulation (cPEI; Davies et al, 2008, Mol Ther 16:1283) induces a mild transient inammatory response characterised by increased neutrophils in bronchoalveolar lavage (BAL) uid. Here we have examined the effect of repeat dosing with cPEI. Two groups of sheep (n=6) were each administered a single dose of cPEI/pCIKLux (32mg DNA at 1.6mg/ml). Expression of luciferase (lux) protein and mRNA were measured either at day 1 (Group 1) or day 15 (Group 2). Mean lux protein expression at day 1 was 19.4 +/- 6.5 RLU/mg lung protein, which fell to background S339
Nonviral gene delivery to the lung is a promising approach for the treatment of a various number of devastating diseases. However, its use has been limited, in part, by the lack of cell specic targeting of DNA. The alveolar epithelial type I (ATI) cell, in particular, is an attractive cell type to target, as it comprises 95% of the internal surface area of the lung. However, ATI-specific gene delivery studies have been limited due to a lack of understanding of the true transcriptional properties of these cells and difculty in isolation of primary cell cultures. To overcome this, we characterized two models of ATI cells, the R3/1 cell line and primary rat alveolar type II cells maintained on plastic for 5 days, to be used for initial gene delivery studies. Immunouorescence analysis has identied these cell types as good models for ATI cells, as evidenced by their strong expression of the ATI markers, T1α and Aquaporin-5, and absent expression of ATII markers, Surfactant Protein B and Lamellar Body Membrane 180. Using these models, we have developed a method for ATI-specic targeting of plasmid DNA through utilization of cellspecic DNA nuclear import sequences. We have previously shown that nuclear import of plasmids in non-dividing cells is mediated by specic sequences of DNA that bind to transcription factors in the cytoplasm. These transcription factors contain nuclear localization signals that can be presented to the importin nuclear import machinery to promote nuclear import. If bound to the transcription factors, the DNA can be carried into the nucleus. To date, we have identied DNA sequences that act in all cell types as well as specic cell types, due to the transcription factors bound. We screened the promoters from several genes previously identied in the literature to be specic for or enriched in ATI cells for the ability to mediate DNA nuclear import in this specic cell type after cytoplasmic microinjection. The promoters of these genes were inserted into a plasmid lacking any other DNA nuclear targeting sequence, and the plasmids were labeled with a triplex-forming Cy3-PNA. Of the sequences examined, we demonstrate that only a DNA sequence within the rat T1α promoter was able to mediate plasmid DNA nuclear import in R3/1 and day 5 primary rat alveolar epithelial cells. When microinjected into other cell types, the T1α promoter displayed no DNA nuclear import activity, suggesting that this sequence acts in a type I cell-specic manner. This highlights a new property of the T1α promoter as well as the ability to directly target the ATI cell over other cell types of the lung.
879. Effects of LPC on Lung Mechanics in NonHuman Primates P. Hiatt, R. McConnell, N. Grove, D. Dang, N. Brunetti-Pierri, D. Palmer, A. Beaudet, P. Ng. Depts. of Pulmonary Pediatrics and Molecular Human Genetics, Baylor College of Medicine, Houston, TX.
Efcient transduction of the airway epithelium and long-term expression of the transgene are essential elements for successful Cystic Fibrosis (CF) gene therapy. Parsons et al (2008) showed that lysophosphatidylcholine (LPC) opens tight junctions between cells allowing improved access to viral receptors on the baso-lateral surface of the airway epithelium in mice. We have previously shown that targeted lobar aerosolization of a mixture of LPC and Helper Dependent Adenovirus (HDAd) into nonhuman primate lungs results in uniform, high level pulmonary transduction. Our goal is to use LPC as a carrier in the delivery of HDAd for gene therapy in CF patients. In this study we investigated the effects of LPC on lung mechanics of non-human primates. Methods: Baseline pulmonary function (PF) in 6 baboons was measured using the Flexivent respiratory mechanics platform (Scireq). Animals were intubated with a cuffed endotracheal Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
tube, placed under general anesthesia, and their lungs were inated to 30cm of H2O prior to each of the PF measurements. Following baseline measurements, LPC was aerosolized in two different concentrations which were previously shown to yield signicant pulmonary transduction by HDAd. Two ml/lobe of 0.1% LPC was delivered into each of the six major lung lobes of baboons using an intracorporeal nebulizing catheter (AeroProbe) inserted into a bronchoscope. PF measurements were taken after LPC administration and every 15 minutes thereafter up to 2.5 hours to determine changes in lung mechanics. A nal PF measurement was taken at 24 hours post LPC administration for those animals with lung functions that did not return to baseline at the end of the 2.5 hour testing time. Three weeks after administration of 0.1% LPC, 0.05% LPC was administered to the same animals and PF measurements were obtained as previously described. Results: Maximal PF changes occurred 15 minutes following LPC administration for both 0.05% and 0.1%LPC. Mean respiratory resistance increased 53%+14%(SE) and 44%+14% above baseline and compliance decreased 25%+3.9% and 27%+7% below baseline after the delivery of 0.05% LPC(n=6) and 0.05%LPC+vector(n=3), respectively. Respiratory resistance in these animals returned to normal in less than 3 hours. When 0.1% LPC(n=5) was administered to the same baboons, their mean respiratory resistance increased 127%+32% over baseline and compliance decreased 35%+4.1% from baseline. When 0.1%LPC+vector was administered to 3 baboons, their mean respiratory resistance increased 338%+108% over baseline and compliance decreased 63%+4.2% from baseline. Pulmonary function changes returned to baseline within 24 hours of LPC+vector administration. No significant changes in FiO2 or SpO2 were observed following administration of either concentrations of LPC. Conclusion: LPC (0.05% and 0.1%) is associated with increased respiratory resistance and decreased compliance measurements when aerosolized to the lung. Maximal changes in PF occur within 15 minutes of delivery. These results indicate that LPC delivered by aerosol to the airways of the lung produce signicant but temporary pulmonary function changes in non-human primates in a dose-dependent manner.
880. Repeat Aerosol Delivery of Concentrated PEI/pDNA to the Sheep Lung
Gerry McLachlan,1 Lee A. Davies,2 Cath M. Gordon,1 Christina Vrettou,1 Eilidh Baker,1 Peter Tennant,1 Alison Baker,1 Rebecca L. Coles,2 Dominique McCormick,2 Stephanie G. Sumner-Jones,2 Stephen C. Hyde,2 Deborah R. Gill,2 D. David S. Collie.2 1 The Roslin Institute & R(D)SVS, University of Edinburgh, Edinburgh, United Kingdom; 2Gene Medicine Group, NDCLS, University of Oxford, Oxford, United Kingdom. Considerable advances have been made in lung-directed gene therapy, however it is unlikely that clinical benet to patients with chronic lung disease can be achieved without repeat administration. For viral vectors, the ability to deliver multiple doses is normally limited by the host immune response, which results in attenuated expression following consecutive exposures. Importantly, aerosol delivery of non-viral gene transfer agents to the lung is not generally associated with the same problem. We have previously demonstrated that aerosol delivery of plasmid DNA (pDNA) complexed with polyethylenimine (PEI) to sheep, either at the standard (low) concentration or with a novel concentrated formulation (cPEI; Davies et al, 2008, Mol Ther 16:1283) induces a mild transient inammatory response characterised by increased neutrophils in bronchoalveolar lavage (BAL) uid. Here we have examined the effect of repeat dosing with cPEI. Two groups of sheep (n=6) were each administered a single dose of cPEI/pCIKLux (32mg DNA at 1.6mg/ml). Expression of luciferase (lux) protein and mRNA were measured either at day 1 (Group 1) or day 15 (Group 2). Mean lux protein expression at day 1 was 19.4 +/- 6.5 RLU/mg lung protein, which fell to background S339